Available Technology
Methods and applications for stitched DNA barcodes
Technology:
"Bar-code fusion genetics" - detecting genetic interactions
Markets Addressed
This new technology (“bar-code fusion genetics” or BFG) has been developed for detecting genetic interactions in S. cerevisiae and other organisms. The BFG method applied to genetic interaction discovery (BFG-GI) exploits existing libraries of strains carrying bar-coded gene deletions, and harnesses the throughput and economy of next-generation sequencing technology. The BFG-GI technology has the potential to allow a single technician in a single year to generate a map of genetic interactions amongst all 18 million S. cerevisiae gene pairs in any given growth condition.
The BFG method also permits dramatically increased efficiency of protein interaction discovery in combination with the HQ-Y2H method (BFG-Y2H), In the first step of BFG, a heterogenous pool of cells is produced such that each cell carries one particular pair of engineered loci (with potentially millions of possible locus pairs being represented in the pool). For BFG-Y2H, each pair of engineered loci consists of a DB-X and AD-Y protein fusion, with DB and AD being the DNA-binding and activation domains of the Gal4 transcription factor, and X and Y being proteins of interest. Protein interaction can reconstitute Gal4 and drive expression of a selectable reporter gene. Strains carrying each DB-X or AD-Y locus are marked (either on a plasmid or in the genome) with an identifying barcode sequence. A DB-X strain pool is mated en masse with an AD-Y strain pool. This pool is selected for the interaction-reporter, and an aliquot of the pool is also counterselected for the AD-Y plasmid. For each strain pool, cells are pelleted, resuspended in the appropriate aqueous reagents, and encapsulated in a water-in-oil emulsion such that no more than 1 cell is present in each emulsion droplet. Cells are lysed, allowing cellular DNA to combine with aqueous reagents without disrupting the emulsion. The pair of barcodes within each encapsulated cell are fused together using crossover PCR which occurs when the emulsion is thermally cycled. DNA is purified, and the abundance of cells for each component strain is identified by parallel sequencing of fused-barcode tags.
Application of the BFG-Y2H technology could allow multiple global interactome maps, each in the context of a different specific genetic background (e.g., addition of a particular activated kinase or other modifying enzyme). In theory, dozens of global interactome maps could be produced by a single technician within one year.
Quantitation of fused-barcode tags
Legend: Black circles show expected absence of a fused barcode between two loci that each have known-defective down-tags. The blue square surrounds a set of negative control gene pairs, in which no off-diagonal counts are expected because deletions for these genes were only present in one mating type. Blue circles show a few visually-identified potential alleviating interactions—none have been previously tested for genetic interaction but one (ADA2/SAC3) corresponds to a known protein interaction. Green, yellow and white circles show a few visually-apparent potential synergistic interactions, which either agree (green) or disagree (yellow) with previous interaction data or have not been previously examined (white). Of the synergistic interactions identified here that had been previously tested, 80% were confirmed. These results show the BFG method can quantify doubly-barcoded strains within a heterogenous pool, reveal the effects of selection, and identify interactions.
Additional Information
Intellectual Property Status: Issued U.S. patent nos.: 8,268,564
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Inventor(s):
Mellor, Joseph C.
Roth, Frederick P.
Suzuki, Yo
Categories:
For further information, please contact:
Grant Zimmermann, Director of Business Development
(617) 495-3067
Reference Harvard Case #2956